Exploring the Intriguing World of Fungal Diversity in the Oral Cavities of a Native Community in Siltepec, Chiapas, Mexico

Abstract

This study explores the relationship between filamentous fungi and dental caries in isolated indigenous communities in Siltepec, Chiapas, Mexico. A total of 37 oral swabs were collected, with 22 participants harboring filamentous fungi, primarily from the genus Cladosporium. Statistical analysis using Student’s t-test and the Mann–Whitney U test revealed a significant reduction in extensive and fully cavitated caries (p < 0.0001) in individuals with fungi, while those without fungi exhibited higher rates of dental decay. Participants with fungi had a higher prevalence of healthy teeth and incipient caries. The findings suggest that traditional maize-based diets, particularly fermented beverages like pozol, may promote the growth of beneficial fungi in the oral microbiome, offering a protective effect against dental caries through microbial competition and the alteration of the oral environment. These results underline the need for further research into the long-term impact of traditional diets on oral health and the potential use of natural substances, such as probiotics and plant-based antimicrobials, to maintain oral homeostasis and prevent caries.

1. Introduction

Fungi are cosmopolitan organisms that colonize diverse natural environments include the oral human cavity. However, its distribution varies according to the region of the world, weather, and specific local environment [1]. The oral cavity is an entry portal for the human body through which air, solids, and liquids pass. The adult oral cavity harbors a dense and diverse indigenous microbiota consisting of protozoa, viruses, bacteria, and fungi. The human oral cavity contains a complex microbiome, estimated to include around 600 bacterial species [2] and 100 fungal species [3]. These indigenous microbes must interact and coevolve with each other and with the host while adapting to diverse and rapidly changing conditions.

While the role of oral bacteria in human health and disease is well understood [4,5,6], the role of oral fungi, except for Candida, remains largely uncharacterized [7]. The establishment of different species of common genera at different ages likely reflects favorable environmental conditions. Changes in the adult oral microbiota are associated with various disease states. The colonization of microorganisms in the oral flora depends on suitable sites and conditions for microbial retention and growth, making bacteria–tissue interactions crucial factors in establishing and maintaining microorganisms in the oral cavity [8,9,10].

The oral microbiome is a complex and dynamic environment composed of a wide variety of microorganisms, including bacteria, fungi, and viruses. These microorganisms interact with each other and the host to maintain homeostasis but can become imbalanced in response to poor oral hygiene, diet, or other health conditions. Recent research has shown that maintaining oral microbiota homeostasis over time is essential to preventing various oral pathologies and ensuring long-term dental health.

Individuals are susceptible to fungal infections, with the Candida genus (spp. albicans, glabrata, parapsilosis, and krusei) being the most common fungi in 20% to 70% of patients. Other species present in the oral cavity include Aspergillus spp., Penicillium spp., Fusarium spp., Aureobasidium spp., Exophiala spp., Eurotium spp., Cladosporium spp., and Saccharomyces spp. [11]. Fungi such as Rhizopus and Fusarium in the oral cavity are normally avirulent in healthy individuals but can cause fatal disseminated infections in patients with suppressed immunity [12,13]. The use of antibiotics, malnutrition, premature birth, and old age are among the most common predisposing factors for opportunistic fungal infections [14].

Fungal species inhabit the oral cavity and play critical roles in both health and disease. However, the oral fungal microbiome (mycobiome) is not well characterized [15]. There is less information available on filamentous fungi in healthy oral cavities. The communities where participants’ samples were taken (Progreso, Miguel Hidalgo, Jose M. Morelos, and Francisco I. Madero) are rural communities located in mountainous areas with at least 95% indigenous populations and a very high degree of marginalization. The communities are small, with fewer than 1000 inhabitants without nearby dental service. Their diet is mainly based on the consumption of maize and derivatives such as sour pozol (fermented corn beverage). Therefore, the aim of this study was to isolate and identify the fungi present in the oral cavity of 37 patients from the rural community of Siltepec in Chiapas, Mexico.

2. Materials and Methods

2.1. Sample Collection

The study was conducted in four isolated indigenous communities in the municipality of Siltepec, Chiapas (Progreso, Miguel Hidalgo, Jose M. Morelos, and Francisco I. Madero). These communities are geographically isolated due to the challenging orographic conditions, with Progreso and Miguel Hidalgo being particularly difficult to access. A total of 145 participants were selected, with ages ranging from 15 to 86 years. Of these participants, 99 were female and 46 male.

Oral swabs were collected to identify the presence of filamentous fungi and assess dental caries. The types of caries assessed included initial caries, moderate caries, extensive caries, and full caries. The average number of initial caries per patient was 6.5, while that of moderate caries was 2.6, extensive caries was 0.87, and full caries was 10.05.

Once the requirements were met, a feeding survey was conducted, and a clinical examination was performed using the ICDAS II method to determine the oral health status of the participating volunteers. Samples were collected from dental plaque on the teeth using polystyrene swabs and transferred to 1.5 µL microtubes, which were stored on ice for transport.

2.2. Fungal Isolation

All fungal strains were isolated from the 37 collected samples. The contents of the polystyrene swabs were dissolved in 2 mL of distilled water and homogenized. Dilutions of 1:100 and 1:1000 were prepared in distilled water. Each diluted suspension was plated on potato dextrose agar using the spread plate technique. The plates were incubated at 25 ± 2 °C for 7 to 10 days.

2.3. Morphological Characterization of Filamentous Fungi

All fungal isolates were incubated in potato dextrose agar (PDA) to observe fruiting bodies and fast sporulation. The identification of fungal species was based on the analysis of macroscopic characteristics related to each strain, including colony texture, color on the front and reverse sides, pigment production, colony size, and growth time. These characteristics were recorded and evaluated for their potential importance in fungal line classification.

Fragments of PDA plates, approximately 1 cm² in size, were cut and placed on glass slides. Each strain was inoculated at both ends of the PDA fragment, covered with a sterile coverslip, and incubated at 25 ± 2 °C until characteristic reproductive structures (approximately 2 weeks) appeared. The coverslips were then removed and placed on slides containing lactophenol cotton blue. A fragment of agar from the glass slide was also detached, and a drop of lactophenol cotton blue was applied to the spot it occupied, followed by placing a new coverslip. The slides were observed under a microscope, and the fungi were identified following the criteria of the Illustrated Genera of Imperfect Fungi manual [16].

2.4. Genetic Characterization

The strains were grown in potato dextrose broth and incubated at 37 °C with shaking at 250 rpm for two to three weeks. The mycelium was washed twice with 1X TE buffer. A portion of the mycelium was transferred to a 1.5 mL conical tube containing glass beads. The conical tubes were then placed at −70 °C for one hour. To each tube, 250 μL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA) was added, and the mixture was homogenized using a cellular breaker for 40 s. Subsequently, 250 μL of lysis buffer and 500 μL of phenol/chloroform/isoamyl alcohol (25:24:1) were added. The DNA was precipitated with 3 M sodium acetate (pH 7) and isopropanol and washed with 70% ethanol. Finally, the DNA was resuspended in nuclease-free water. DNA concentration and purity were analyzed using a Nanodrop spectrophotometer.

Amplification of a partial 28S ribosomal gene was performed using the primers LS1 (5′-AGTACCCGCTGAACTTAAG-3′) and LR5 (5′-CCTGAGGGAAACTTCG-3′). The amplification conditions used have already been described [17].

The amplification products were purified using the Gel DNA Recovery Kit from ZYMO RESEARCH following the manufacturer’s instructions. Sequencing was performed at the Molecular Biology Unit, Institute of Cellular Physiology, National Autonomous University of Mexico (UNAM).

3. Results

3.1. Fungal Isolates

The presence of filamentous fungal growth was only present in 37 samples. The results revealed the presence of 40 yeasts and 22 filamentous fungi among the 37 analyzed samples. In some samples, it was not possible to isolate any fungus. Most patients had one fungus, while only seven presented two or more. Among the samples, 22 (59.4%) showed no fungal growth, 5 (13.5%) had no yeast growth, and 3 (8.1%) did not exhibit any growth. Thirteen fungi appeared to excrete an exoenzyme.

The comparison between patients with and without filamentous fungi in terms of dental caries revealed significant differences in both the total caries count and distribution. A total of 22 samples were identified with filamentous fungi, of which 10 came from the community of Progreso and 12 from Miguel Hidalgo, both geographically isolated due to challenging orographic conditions.

The comparison between patients with and without filamentous fungi showed significant differences in the incidence of dental caries. Patients without filamentous fungi exhibited a higher incidence of fully cavitated caries and extensive caries. In contrast, the group with filamentous fungi showed a higher average of healthy teeth and incipient caries (Figure 1).

Using Student’s t-test, a statistically significant difference in the mean number of dental caries was observed between the two groups (t = −7.82, p < 0.0001). Patients without filamentous fungi had a higher mean number of caries (mean = 10.06, SD = 7.02) compared to those with filamentous fungi (mean = 2.45, SD = 3.46). Similarly, the Mann–Whitney U test confirmed a significant difference in the distribution of caries between the groups (U = 494.0, p < 0.000002). This result supports the finding that patients without filamentous fungi tend to have a higher caries burden compared to those with filamentous fungi. These findings suggest that the presence of filamentous fungi may be associated with a lower incidence of dental caries, though further research is necessary to explore potential causal mechanisms.

3.2. Morphological Characterization of Filamentous Fungi

The morphological colonial characteristics are presented in

Table 1. Macroscopic characteristics of filamentous fungi.

Isolate	Texture	Colonial Form	Elevation	Border	Front Color	Down Color
OP-FV-27a	Fluffy	Circular	Umbilical	Regular	Greenish	Greenish
OP-FV-27b	Plane	Circular	Convex	Regular	Dark gray	Grayish
OP-FV-38	Fluffy	Circular	Plane	Filamentous	White	Greenish
OP-FV-46	Fluffy	Circular	Convex	Regular	Greenish	Dark green
OP-FV-53	Fluffy	Circular	Umbilical	Ragged	Dark green	Dark green
OP-FV-64a	Fluffy	Circular	Convex	Regular	Dark green	Dark green
OP-FV-64b	Fluffy	Circular	Convex	Filamentous	Dark green	Dark brown
OP-FV-66	Plane	Circular	Convex	Filamentous	Dark green	Dark green
OP-FV-68	Plane	Circular	Convex	Regular	Brownish	Opaque green
OP-FV-69a	Fluffy	Circular	Umbilical	Regular	Dark green	White-green
OP-FV-69b	Fluffy	Circular	Umbilical	Regular	Dark green	Light black
OP-FV-89	Fluffy	Circular	Umbilical	Filamentous	Dark gray	Brown-grayish
OP-FV-98a	Plane	Circular	Convex	Regular	Greenish	Dark green
OP-FV-98b	Fluffy	Circular	Umbilical	Regular	Dark green	Greenish
OP-FV-99	Fluffy	Circular	Umbilical	Filamentous	Dark green	Dark green
OP-FV-141a	Fluffy	Circular	Umbilical	Regular	Black	Black
OP-FV-141b	Fluffy	Circular	Umbilical	Regular	Dark green	Dark green
OP-FV-148a	Plane	Circular	Convex	Regular	Gray-greenish	Dark green
OP-FV-148b	Fluffy	Circular	Umbilical	Regular	Dark green	Light green
OP-FV-157a	Fluffy	Circular	Umbilical	Regular	Dark green	Dark green
OP-FV-157b	Fluffy	Circular	Convex	Regular	Pink	Pinkish
OP-FV-157c	Fluffy	Circular	Umbilical	Regular	Gray-greenish	Dark green

. All isolates exhibited slow growth (10–20 mm in 18 days). Fungi have differences between them in texture, form, elevation, border, and color (Figure 2).

Microscopically, all fungi displayed thin, septate, branched hyphae that were hyaline to brown. Most isolates produced erect, dark, septate hyphae. Conidiophores were also darkly pigmented, often septate, and showed tree-like branching (Figure 3). Fragile chains of dematiaceous conidia were produced, with a dark hila or scar at their point of attachment to the conidiophore or other conidia. Chains of conidia easily disarticulated, and based on these characteristics, we identified these fungi as Cladosporium sp. Some other isolates presented smooth-walled, uncolored to pale brown conidiophores. The conidial heads (vesicles) consisted of uniseriate phialides and were loosely columnar. Conidia (spores) were found in chains, ranging from spherical to ellipsoidal. These isolates were identified as Eurotium sp. Macroconidia were several-celled, slightly curved, or bent at the pointed ends and typically canoe-shaped, indicating Fusarium sp. The conidiophores were branched near the apex and penicillate and ended in a group of phialides, while the conidia were hyaline in dry basipetal chains, suggesting Penicillium sp. Dark conidiophores, mostly simple and dark conidia, typically with both cross and longitudinal septa, and various shapes indicated Alternaria sp. However, it was not possible to identify all isolated fungi.

3.3. Genetic Characterization

The PCR products (Figure 4) from 13 strains isolated from 11 patients in the community of Siltepec, Chiapas, Mexico, ranged from 577 to 1652 bp for the 28S gene. BLAST analysis conducted in NCBI revealed a high identity of the 28S gene sequences with Penicillium, Acremonium, Alternaria, and Cladosporium (Figure 5). Candida spp. were found in a higher percentage of patients (86.5%). Thirteen fungi were genetically identified, with Cladosporium spp. (69.2%), Alternaria sp. (15.4%), and Penicillium and Acremonium (7.7%) being the most prevalent (

Table 2. Fungal species in oral mycobiome of healthy individuals.

	Length	Enzyme Production	Identification	% Identity
OP-FV-157a	590	yes	Cladosporium cladosporioides	98
OP-FV-157b	1279	yes	Acremonium charticola	99.19
OP-FV-157c	896	yes	Cladosporium endophytica	96.33
OP-FV-69a	903	yes	Cladosporium cladosporioides	100
OP-FV-64a	895	yes	Cladosporium sphaerospermum	99.5
OP-FV-98a	875	yes	Alternaria alternata	95.79
OP-FV-98b	896	yes	Cladosporium sphaerospermum	98.67
OP-FV-89	1652	yes	Cladosporium cladosporioides	99.77
OP-FV-68	767	yes	Cladosporium cladosporioides	99.41
OP-FV-27b	1568	no	Penicillium citrinum	100
OP-FV-66	577	no	Cladosporium cladosporioides	98.81
OP-FV-53	909	no	Alternaria alternata	99.45
OP-FV-38	1558	no	Cladosporium cladosporioides	97.82

). Notably, all filamentous fungi belonged to the phylum Ascomycota.

3.4. Statistical Analysis

The statistical analysis was performed with Jamovi version 2.3.28. Descriptive statistics, t-tests, and Mann–Whitney U tests were applied to compare caries prevalence between groups. The averages and standard deviations for initial, moderate, extensive, and fully cavitated caries were calculated.

4. Discussion

The oral cavity, as the initial entry point for food and drink, is a dynamic environment that provides a habitat for a diverse range of microorganisms, including protozoa, viruses, bacteria, and fungi. These microorganisms co-exist with each other and with the host, adapting to fluctuating conditions such as nutrient availability, pH changes, and environmental exposure. While these microorganisms generally maintain a balanced state, any disruption, such as poor oral hygiene or health complications, can lead to imbalances, favoring the growth of pathogenic species. The microbial interactions within the oral cavity are complex, with bacteria often receiving the most attention, but this study highlights the significant role of fungi in oral health, particularly in rural, isolated communities [3].

In this study, we examined 37 samples from individuals in the Siltepec community and demonstrated the presence of 22 culturable and 22 non-culturable filamentous fungi. This is in line with other research that has also identified the presence of both culturable and non-culturable fungi in the oral cavity. However, the number of species isolated in our study is lower than that reported by Ezikanyi et al. [3], who identified 74 culturable fungal genera from just 20 individuals. This discrepancy could be attributed to several factors, including differences in geographical location, diet, oral hygiene practices, and environmental exposure. The isolation of 13 exoenzyme-producing fungi in our study, the majority of which belong to the genus Cladosporium (e.g., Cladosporium cladosporioides, C. endophytica, C. sphaerospermum), suggests that these fungi may play a crucial role in the oral cavity. The production of exoenzymes could help fungi adapt to the nutrient-rich environment of the mouth, allowing them to break down complex molecules for energy. These enzymes might also contribute to the fungi’s ability to inhibit the growth of bacteria, particularly Gram-positive species such as Streptococcus mutans, a common cause of dental caries [18]. This microbial competition could explain the significant differences in dental health between individuals with and without filamentous fungi in their oral microbiome.

The genus Cladosporium has been reported in several studies as one of the dominant fungal species in the oral cavity [3]. Its ability to thrive in diverse environments, including soil, air, and water, as well as its frequent occurrence in food, supports the idea that it could easily colonize the human oral cavity. Interestingly, our study found fewer species of Aspergillus, a genus commonly associated with oral health issues, compared to other studies [7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]. This could suggest that Cladosporium spp. is outcompeting other fungal species in this specific population, possibly due to environmental or dietary factors unique to the rural, indigenous communities studied. The connection between diet and the composition of the oral microbiome is particularly intriguing in this context. The communities of Progreso and Miguel Hidalgo rely heavily on maize as a dietary staple, with fermented maize-based products like pozol playing a significant role in their daily nutrition. Pozol, a traditional fermented beverage, contains a variety of microorganisms that could influence the composition of the oral microbiome. Fermentation processes are known to introduce beneficial microbes and bioactive compounds, which may suppress the growth of cariogenic bacteria [22,23,24,25,26]. The regular consumption of these fermented products could promote the growth of fungi like Cladosporium spp., which may provide a protective effect against dental caries. This hypothesis is supported by the lower incidence of extensive and fully cavitated caries in individuals with filamentous fungi in their oral microbiota.

Additionally, the fermentation process associated with maize products like pozol may alter the pH of the oral cavity, creating an environment less favorable for the growth of cariogenic bacteria. Fermentation produces organic acids and other metabolites that can modify the oral environment, promoting microbial diversity and inhibiting the proliferation of harmful species. The protective role of fungi in this context is further supported by Student’s t-test and the Mann–Whitney U test, which demonstrate statistically significant differences in dental caries between individuals with and without filamentous fungi.

The geographical and socio-environmental isolation of the communities in Progreso and Miguel Hidalgo further compounds these findings. Isolated populations are less exposed to external pathogens and may have a unique oral microbiome shaped by traditional dietary practices. The combination of dietary factors, limited exposure to industrialized food products, and reliance on locally sourced, naturally fermented foods could be key in understanding the lower prevalence of caries in these communities [3]. Traditional diets, rich in maize and its derivatives, appear to play a role in fostering a microbiome that protects against dental decay. This contrasts with urban populations, where the prevalence of sugary, processed foods contributes to higher rates of dental caries.

However, it is important to acknowledge the limitations of this study. The cross-sectional nature of the study limits our ability to draw definitive conclusions about causality. While we observed significant associations between the presence of filamentous fungi and reduced caries, longitudinal studies are necessary to determine whether these fungi play a direct role in protecting against dental decay. Additionally, the relatively small sample size may reduce the generalizability of the findings. Future research should aim to explore the specific interactions between fungi, bacteria, and diet in larger, more diverse populations [22,23,24,25,26]. The use of natural substances, particularly plant-based antimicrobials and probiotics, could be a promising area of research for oral health interventions. These natural compounds, found in fermented foods and traditional diets, could help maintain oral homeostasis and prevent the overgrowth of cariogenic bacteria [21].

Butera et al. [27] conducted an insightful study that focused on the characterization of the fungal microbiota in the oral cavity, particularly in patients with peri-implantitis, a condition involving inflammation around dental implants. Their work found a significant presence of Candida species and other fungal genera such as Aspergillus and Penicillium in their samples, with notable differences in fungal composition between healthy and diseased individuals. This complements our findings in a rural, indigenous population, as we also identified Candida and filamentous fungi like Cladosporium and Penicillium as dominant genera.

However, while Butera et al.‘s [27] study highlighted the role of fungi in a pathological setting, our work extends the scope by exploring the potential protective effects of these fungi in preventing dental caries, particularly in a population with unique dietary habits that include fermented maize products like pozol. The overlap in fungal species between both studies underscores the importance of further investigating the complex interactions between fungal communities, diet, and oral health. Moreover, the stark geographical and environmental differences between the populations studied in both works offer a valuable opportunity to explore how local factors influence the composition and role of the oral mycobiome. Future research could build on both studies to assess whether dietary practices in indigenous communities confer a protective effect against fungal overgrowth or other oral diseases, as suggested by the lower incidence of dental caries in our study population. This added perspective reinforces the need for a broader understanding of fungal communities in both health and disease, highlighting the intersection between microbial ecology, diet, and oral health as a promising area for future research in rural and indigenous populations, where access to dental care is often limited, such interventions could provide a cost-effective, minimally invasive means of improving oral health outcomes. This study underscores the complex interplay between diet, the oral microbiome, and dental health in isolated indigenous communities. The presence of filamentous fungi, likely influenced by traditional dietary practices, appears to offer a protective effect against dental caries. Future studies should focus on the role of natural substances and probiotics in maintaining oral health and further investigate the long-term impact of dietary habits, such as the consumption of fermented maize products like pozol, on the oral microbiome.

5. Conclusions

This study identified the mycobiome present in the oral cavity of healthy individuals from an indigenous group in Siltepec, Chiapas, a region that had not been previously explored in this context. Our results showed that most participants exhibited few dental caries, suggesting a potential protective role of the local oral fungi, specifically filamentous species such as Cladosporium and Penicillium. The complex relationship between the development of oral microbial flora and factors such as diet, nutritional habits, and local environmental conditions was evident in this indigenous community, particularly with the consumption of fermented maize products like pozol.

These findings align with previous studies, such as Butera et al. (2021) [27], which identified a significant presence of Candida and Aspergillus in individuals with peri-implantitis, highlighting how fungi can play both protective and pathogenic roles depending on the oral health context. While Butera’s work [27] focused on diseased oral environments, our study suggests that in healthy, rural populations, filamentous fungi may contribute to oral health by preventing the overgrowth of cariogenic bacteria.

Our study demonstrates that traditional dietary practices may influence the oral mycobiome in ways that are beneficial for dental health, as evidenced by the lower incidence of fully cavitated caries. This unique intersection between diet, microbial ecology, and oral health offers promising insights for future preventive strategies. Further research could expand on these dynamics, particularly exploring how fermented maize products and the geographical isolation of indigenous communities shape their oral microbial environments. This knowledge could inform the development of oral health interventions that leverage local diets and microbiomes to maintain or improve dental health in similar populations.